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Print This Article PEER-REVIEWED ARTICLE bioresources.com The Study of Glucose and Xylose Content by Acid Hydrolysis of Ash Wood (Fraxinus excelsior L.) after Thermal Modification in Nitrogen by HPLC Method Jakub Gawron,a Andrzej Antczak,b,* Sławomir Borysiak,c Janusz Zawadzki,b and Adam Kupczyk a This study aimed to determine glucose and xylose content by acid hydrolysis of wood samples, both unmodified and thermally modified (modification time was 2, 6, 10 hours), using high performance liquid chromatography. Optimization of the hydrolysis process on the native ash wood samples showed that 3 h was the best time in the hydrolysis process. After that time, 58.8% of glucose and 20.8% of xylose were obtained. In turn, chromatographic analysis showed incomplete hydrolysis of ash wood samples, which were modified in a nitrogen atmosphere, especially at shorter times (2 and 6 h) of modification. With longer modification times (10 h), the hydrolysis of ash wood samples was completed. The above mentioned problem was caused mainly by the increase of cellulose crystallinity degree. The decrease of this parameter was observed only after 10 h of thermal modification, which would facilitate the process of acidic hydrolysis. Additionally, it was observed that the thermal modification of ash wood at 190 °C in a nitrogen atmosphere for 10 h caused a drastic decrease in the xylose content (from 20.8% to 8.0%) and only a slight decrease in the glucose content (from 58.8% to 54.9%). Keywords: Acidic hydrolysis; Thermal modification; Ash wood; HPLC; Glucose and xylose content Contact information: a: Faculty of Production Engineering, Warsaw University of Life Sciences – SGGW, 164 Nowoursynowska St., 02-787 Warsaw, Poland; b: Faculty of Wood Technology, Warsaw University of Life Sciences, 159 Nowoursynowska St., 02-776 Warsaw, Poland; c: Faculty of Chemical Technology, Poznan University of Technology, 2 M. Sklodowskiej-Curie Sq., 60-965 Poznan, Poland; * Corresponding author: [email protected] INTRODUCTION Thermal treatment is an alternative to chemical treatment in wood preservation and has been used to some extent for improving timber quality. One way to improve its properties is thermal modification under nitrogen. This type of modification has been successfully introduced and is being implemented on a fairly large scale in France (Vernois 2001). Among the benefits associated with this technique is an improvement in dimensional stability (Kamdem et al. 2000; Rapp and Sailer 2001; Awoyemi and Jones 2011). Additionally, the strength properties of wood are changed, the bending strength decreases, but the modulus of elasticity remains at a similar level (Viitaniemi and Jämsä 1996; Kubojima et al. 2000). Thermal modification changes the colour of wood. This applies to the entire cross-section of wood and the colour is confusingly similar to some species of tropical wood. The darkening of the wood becomes more intensive with increasing time and temperature of thermal modification (Sundqvist 2002; Bekhta and Niemz 2003). It is believed that the main cause of the wood color change under the Gawron et al. (2014). “Hydrolysate content of ash,” BioResources 9(2), 3197-3210. 3197 PEER-REVIEWED ARTICLE bioresources.com influence of thermal modification is the formation of colored degradation products from extractives and hemicelluloses (Sundqvist and Morén 2002; Sundqvist 2004). Additionally, the formation of oxidation products (e.g. quinones) is also considered as the reason for color change (Mitsui et al. 2001; Bekhta and Niemz 2003). Research studies on thermal modification are still ongoing; there are various conditions of modification and methods to analyze modified wood (Zaman et al. 2000; Alén et al. 2002; Sivonen et al. 2002; Nuopponen et al. 2003, 2004; Wikberg and Maunu 2004). Thermal wood modification is typically conducted within a temperature range from 160 °C to 260 °C under conditions of limited supply of oxygen. Currently, several thermal modification technologies in various conditions of gas atmosphere (vacuum, steam, nitrogen, combustion gases) and also by using an oil were developed (Hill 2006; Esteves and Pereira 2009). During thermal modification, large changes occur in the chemical structure of the wood compounds. In particular, there is a degradation of amorphous carbohydrates, mainly hemicelluloses (Rowell et al. 2002; Udaka and Furuno 2003; Boonstra and Tjeerdsma 2006; Gawron et al. 2011). Depending on the conditions and the environment of thermal modification, the amount of carbohydrates decreases. The content of polysaccharides in the thermally modified wood can be determined by hydrolysis and high performance liquid chromatography (HPLC) methods. The hydrolysis process is a reaction that allows the conversion of polysaccharides to monosaccharides. Concentrated and dilute acids or specific enzymes are used for obtaining simple sugars from the polysaccharides (Wyman et al. 2005; Carvalheiro et al. 2008). Mineral acids such as sulfuric, hydrochloric, phosphoric acids, and organic trifluoroacetic acid are the most commonly used. The advantages of the use of acids are their low price and minimal consumption. In turn, the enzymatic hydrolysis process uses highly specialized enzymes. In the case of cellulose three enzymes are used, which act synergistically: endocellulases (hydrolyzing β glycosidic bond within the cellulose chain), exocellulases (splitting off the cellobiose molecule from the end of the chain), and β-glucosidase (decomposing cellobiose molecule to glucose). On the other hand, the enzymes for the hydrolysis of hemicelluloses are much more complex and include endo-1,4-β-D-xylanases, exo-1,4-β- D-xylosidases, endo-1,4-β-D-mannanases, β-mannosidases, acetyl xylan esterases, α- glucuronidases, α-L-arabinofuranosidases, and α-galactosidases (Jorgensen et al. 2003). A number of different analytical methods can be used to separate and analyze sugars, such as thin layer chromatography, gas, liquid chromatography, or capillary electrophoresis. Among the instrumental methods, HPLC is a technique often used in the analysis of sugars (Agblevor et al. 2004, 2007; De Muynck et al. 2006; Rybak- Chmielewska 2007; Rahman et al. 2008; Salman et al. 2011). This method has many advantages; it has high accuracy, the sample requirement is very low (about tens to hundreds of mg), and there is no need for sample derivatization. In the available literature, there is information regarding the use of the HPLC method for analysis of sugars in wood subjected to thermal modification (Inari et al. 2007; Karlsson et al. 2012; Cademartori et al. 2013). But unfortunately, there is no information about the use of this method to examine the content of sugars in ash wood. The ash wood is an important species, because it is often used for thermal modification. Therefore, in this case, from a scientific and practical point of view, the use of the HPLC method is interesting and worthy of attention. Gawron et al. (2014). “Hydrolysate content of ash,” BioResources 9(2), 3197-3210. 3198 PEER-REVIEWED ARTICLE bioresources.com In this work, an attempt was made using HPLC to examine the chemical composition of the main saccharides (xylose and glucose) obtained from ash wood (Fraxinus excelsior L.) unmodified and thermally modified under nitrogen. EXPERIMENTAL Materials The thermal modification was carried out on the ash wood (Fraxinus excelsior L.). The age of the tree was about 80 years. Uncoloured wood taken from the heartwood was used. The average density of the tested ash wood was 731.6 kg/m3 and the average standard deviation 40.4 kg/m3. Methods Thermal modification in nitrogen atmosphere Thermal modification in nitrogen atmosphere was carried out in three-necked round bottom flask (250 cm3) placed in an oil bath. In the modification process, ash wood ground to the form of sawdust (fraction 0.43 to 1.02 mm) was used. Duration of the respective modification process was 2, 6, and 10 h at 190 °C. The modification was preceded by drying the samples to an absolutely dry state. Then, the temperature was raised to 130 °C at the rate of 1 °C/min, and nitrogen with a purity of 99% was supplied to the flask from a cylinder. Nitrogen flow was maintained at a constant rate throughout the thermal modification process, including the cooling of samples. Temperature was maintained at 130 °C for 30 min. In the next stage, the temperature in the flask was raised to 190 °C at the rate of 1 °C/min. When the temperature in the flask reached 190 °C, the appropriate thermal modification occurred, which lasted for 2, 6, or 10 h. After the specified time, the samples were cooled for 30 min to ambient temperature and then placed in a desiccator. The samples, which were taken after the modification process, were subsequently subjected to hydrolysis, in order to determine the content of the saccharides (xylose and glucose) in the tested ash wood by HPLC. Hydrolysis of wood samples Acid hydrolysis (Kačik and Solár 1999; Antczak et al. 2012) was performed for ash wood samples unmodified and thermally modified under nitrogen at 190 °C for 2, 6, and 10 h. Each wood sample was then subjected to a second series of hydrolysis. In the hydrolysis process, finely ground wood in the form of sawdust (fraction below 0.43 mm) was used. At the beginning, wood dust was extracted with a mixture of chloroform (Chempur, Poland) and 96% ethanol (Chempur, Poland) in a weight ratio of 93:7. It was then dried to constant weight in a vacuum drier at 60 °C and under pressure 0.4 kPa. After drying, the extracted wood samples (approximately 100 mg) were placed in flasks with a capacity of 100 cm3, which were poured with 1 cm3 of 72% sulfuric acid (Chempur, Poland), and tightly stoppered. Then, samples were heated at 30 °C for a period of 1 h, cooled to room temperature, and 28 cm3 of distilled water was added to each sample. After connecting to a reflux condenser, the flasks were heated at 120 °C in an oil bath.
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